目前研究认为趋化因子与靶细胞表面的受体结合可趋化免疫细胞至相应部位参与感染、炎症、自身免疫性疾病、肿瘤、血管形成等病理生理过程,此外,趋化因子受体的表达影响着免疫细胞的功能,趋化因子也可直接参与抗细菌、抗真菌等众多病理过程.CXC型趋化因子受体CXCR3是现各领域研究热点,它是干扰素诱导单核因子(Mig)、干扰素诱导蛋白10(IP-10)和干扰素诱导T细胞α化学趋化因子(I-TAC)的特异性受体.本文将综述CXCR3-CXCL9/10/11在多种疾病发病机制中的作用.

The mechanism of innate and adaptive immune responses to chronic infections with hepatotropic viruses(HBV,HCV)was studied in 2018.Its mechanism elucidated the dysregulation of natural killer(NK)cells,monocytes,B cells and T cells.In addition,a new target for immune regulation of HBV infection(TLR3/OX40L)was introduced.The discovery of new NK cell immune checkpoints,the involvement of mononuclear macrophages in liver failure and inflammation,sex hormone affecting intrahepatic-resistant bacterial infection through the regulation of humoral immunity,and the communication mechanism between liver and other immune organs have enriched people's understanding of liver immunology and its clinical significance.

肝脏是人体重要的代谢和免疫器官,具有合成、代谢、排泄、解毒和免疫等多种功能.由各种原因导致的肝炎、肝硬化和肝脏肿瘤等肝脏疾病是一类严重危害人民健康的重要疾病.因此,加强对肝脏免疫学的研究,不仅有助于理解肝脏在生理和病理状态下的功能变化,也有助于对肝脏疾病防治药物及策略的研发.随着科学技术的进步,肝脏免疫学已成为多学科合作研究的热点并取得了巨大的进展.

Liver sinusoidal endothelial cells are a major group of nonparenchymal cells in the liver and are involved in immunological surveillance of the liver through the expression of various scavenger receptors and pattern recognition receptors.However,in case of several physiological states,viral infections,and tumor environment,liver sinusoidal endothelial cells maintain immune tolerance in the liver through various mechanisms and cause persistent viral infection and tumor metastasis.This article reviews the mechanisms of immune tolerance ofCD4 + T cells and CD8 + T cells in the liver induced by liver sinusoidal endothelial cells.

Liver sinusoid endothelial cells are the first defense mechanism that protects the liver against various injuries, and they also play a significant role in the development of liver fibrosis and liver cirrhosis caused by chronic liver injury. They are involved in liver fibrosis by mediating liver inflammation, participating in sinusoid capillarization and revascularization, activating hepatic stellate cells, secreting many proinflammatory cytokines, participating in extracellular matrix formation, and mediating liver microcirculation disturbance. Clarification of these mechanisms helps to identify new targets and develop new regimens for the treatment of liver fibrosis.

NOD1 and NOD2 are two intracellular pattern recognition receptors.They sense the major component of bacterial cell walls and their degradated products,then mediate NF-κB and MAPKs signaling pathways to produce the effector molecules involved in the antipathogenic immune response.Furthermore,it has been found in the recent years that NOD1 induces the production of type I interferon through ISGF3 signaling pathways and that NOD2 could recognise virus RNA and activate the MAVs-IRF3 signaling pathways to produce type I interferon.Type Ⅰ interferon could also positively regulate NOD1 and NOD2 functional expression.There-fore,NOD1 and NOD2 induce to large amounts of interferon to mediate innate immune antiviral responses by the aforementioned signaling pathways.Collectively,further understanding the antiviral immune responses mediated by NOD1 and NOD2 may provide new opportunities and strategies for the prevention and treatment of viral infections.

目的:建立干扰素-γ(interferon-γ,IFN-γ)基因敲除小鼠慢性乙型肝炎病-毒(hepatitis B virus,HBV)复制模型.方法:IFN-γ基因敲除(IFN-γ-/-)小鼠繁育并抽提组织DNA进行聚合酶链反应(PCR)及凝胶电泳鉴定基因型.IFN-γ-/-小鼠纯合子9只与野生型C57BL/6小鼠9只同时高压水注射pAAV/HBVl.2质粒,按既定时间点采血检测乙型肝炎表面抗原(hepatitis B virus surface antigen,HBsAg)、乙型肝炎e抗原(hepatitis B virus e antigen,HBeAg)和HBV DNA.血清HBsAg和HBeAg表达水平由电化学发光法进行定量检测.经抽提血清总DNA后,血清HBV DNA由定量PCR进行检测.结果:本实验室繁殖的IFN-γ-/-小鼠均为纯合子基因型.IFN-γ-/小鼠和野生型C57BL/6小鼠血清中HBsAg、HBeAg和HBV DNA持续存在,转染后第40天仍阳性.但是,IFN-γ-/-小鼠血清HBsAg表达水平高于C57BL/6野生小鼠(40天时,P=0.042); IFN-γ-/-小鼠血清HBV DNA持续高水平复制,明显高于C57BL/6野生小鼠(第25天时,P=0.012; 第40天时,P=0.039).两组小鼠血清HBeAg表达水平无差异.结论:IFN-γ-/-小鼠慢性HBV复制模型成功建立,并揭示了IFN-γ在慢性HBV感染中可抑制HBV复制.

Objective To optimize the amplification and sequencing of non structural protein 3 (NS3) gene in patients infected with hepatitis C virus (HCV) genotype 1b. Methods The genotyping after extracting HCV RNA from the serum was detected with type specific primers assay. After designing three pairs of outside primers and three pairs of inner primers, HCV NS3 (HCV genotype 1b) were amplified by a nested PCR assay (option I) with above primers and sequenced. In order to optimize effects of NS3 amplification and sequencing and to reduce the complicated operation, we improved the new nested PCR assay (option II) and redesigned the primers, including a pair of outside primers and a pair of inner primers. We amplified directly HCV NS3 (HCV genotype 1b) with improved nested PCR assay and sequenced NS3 with three forward primers and a reverse primer. Results Compared to the original nested PCR assay (option I), the improved nested PCR assay (option II) is simpler in operation and of higher efficiency. Conclusion The improved nested PCR assay (option II) is more effective and practical for amplification and sequencing of HCV NS3.

Objective To establish a HBV infection model of CXCR3 knocked-out mice.Methods After CXCR3 knocked-out,nine of them and nine wild-type C57BL/6 mice were injected hydrodynamically ten micrograms of pAAV/HBV1.2 DNA into the tail veins.Then,the mice were regularly bled to monitor the serum levels of HBsAg,HbeAg and HBV DNA.The HBcAg in the livers from injected mice were detected by immunohistochemistry.Results All the bred CXCR3 knocked-out mice were homozygous.The serum levels of HBsAg from the CXCR3 knocked-out mice and the wild type C57BL/6 were 1134.69±244.42 and 1759.63±881.20(P=0.096) at the first day after the injection of the pAAVHBV1.2 plasmids;5305.29±1395.06 and 7493.29±658.63(P=0.003) at the fourth day,1615.04±1187.16 and 1536.19±1046.02(P=0.905) at the fifteenth day,and 229.45±79.27 and 228.19±295.02(P=0.996) at fortieth day,respectively;the serum levels of HBeAg from them were 6.65±1.50 and 20.61±4.03(P=0.000) at the first day,6.33±1.61 and 9.79±2.31(P=0.007) at the fourth day,3.52±1.97 and 2.85±0.74(P=0.425) at the fifteenth day,and 1.28±0.06 and 1.90±1.01(P=0.431)at fortieth day,respectively and the serum levels of HBV DNA from them were 4.38±0.22 lgcopies/ml and 6.56±0.16 lgcopies/ml(P=0.008) at day10,4.41±0.88 lg copies/ml and 5.69±0.04 lg copies/ml(P=0.177) at day15,4.48±0.04 lgcopies/ml and 6.44±0.16 lgcopies/m(P=0.004) at day25,and 3.66±0.45 lgcopies/ml and 5.20±0.28 lg copies/ml(P=0.055) at day40,respectively;HbsAg,HbeAg and HBV DNA in CXCR3 knocked-out mice were continuously positive untill the day 40 after the injection of the pAAV/HBV1.2 DNA;Both cytoplasmic and nuclear HBcAg were detected in the livers of the CXCR3 knocked-out mice at the day 4,15,40 after the infection.The positive serumHBsAg,HbeAg and HBV DNA in CXCR3 knocked-out mice were similar to those in the wild type C57BL/6 mice.Conclusion We had successfully established a HBV infection model in CXCR3 knocked-out mouse,which might be helpful way to investigate the relationship between the CXCR3 and its ligands to the HBV infection.

Objective To investigate the inhibition on woodchuck hepatitis virus(WHV) X gene expression by RNA interference.Methods WHV X gene was amplified and restricted with EcoR I and Pst I,then inserted into the EcoR I-Pst I site of pXF3H vector,which finally generated a plasmid expressing WHX-HA fusion protein(pXF3H-WHx).Two plasmids(psiWx1 and psiWx2) expressing siRNA targeting against WHV X gene were constructed by directly inserting the synthesized siRNA templates to the Bgl Ⅱ and Hind Ⅲ sites of psiRNA vector.Hela cells were cotransfected with pXF3H-WHx and psiWx1 or psiWx2,and the expression level of WHx-HA fusion protein was investigated.Results WHX-HA fusion protein expression was inhibited over 90% with siRNA transcribed from psiWx1 dose-dependently,while two nucleotides mutation in the target sequence thoroughly abrogated the RNAi effect.Conclusion RNAi could inhibit the WHV X protein expression sequence-specifically and dose-dependently.

乙型肝炎表面抗原(HBsAg)变异可导致抗原性改变.HBsAg a决定簇最常见的变异位点是G145R.G145R变异可能改变原来的抗原表位,并形成新的抗原表位.本研究探讨G145R HBsAg能否刺激机体产生抗体及免疫应答的情况。

Objective To construct the sH-2Dd-HBs complex. Methods β2m gene and the extracellular region H-2Dd were amplified from the spleen cellisolation of BALB/c mice by reverse transcriptase-PCR, and a 15-amino acid substrate peptide for BirA-dependent biotinylation was added to the COOH-terminus of H-2D heavy chain. The products were subcloned into pET-28a ( + ) vector, and the fusion proteins were then expressed via induction and purified to a high purity. U-sing sH-2Dd -BSP as heavy chain and β2m as light chain, thery were refolded in the presence of an H-2D restricted antigenic peptide, and the sH-2D -HBs complex was formed. Results Direct sequencing results showed that the homologies of nucleotides between presumed and the sequence obtained were 100%. The H-2Dd-BSP and β2m fusion proteins were successfully expressed and rena-tured in the presence of an antigenic peptide ( HBs201-209aa),Conclusion Obtaining of sH-2Dd-HBs complex lays the foundation for further construction of tetrameric H-2D -HBs complexes for the research of the mechanisms of specific CTL responding and effecting.